Project Summary Persistent nucleic acid structures containing RNA-DNA hybrids with a displaced single- stranded DNA (R-loops) lead to DNA damage including double strand breaks (DSBs), replication stress, and genomic instability. The overall goal of this research proposal is to reveal; i) the interplay of the 5'-3'-exoribonuclease 2 (XRN2) and poly(ADP-ribose) polymerase 1 (PARP1) in preserving genomic integrity via resolving R-loops, and preventing replication stress/DNA damage, and ii) define the mechanism of synthetic lethality induced by their concurrent deficiencies to understand the translational implications of XRN2-related vulnerabilities. The XRN2 protein degrades downstream-cleaved RNAPII-associated RNA to resolve R-loops and promotes transcription termination. It is also involved in general RNA degradation, gene silencing, and rRNA maturation. Overall, XRN2's role is well understood in RNA metabolism, however, its impact on other cellular processes, especially in genome maintenance is poorly understood. Our previous study provided evidence that XRN2 loss leads to elevated R-loop and DSB formation consequently generating genomic instability in the human fibroblast as well as in cancer cells. However, there is a lack of functional information regarding its specific contribution in resolving replication stress, coordinating DNA repair, and promoting cell survival. Furthermore, genetic alterations in XRN2 including mutation, copy number variation, and mRNA expression are frequent in a variety of cancers. Also, XRN2 polymorphism has been linked to increased risk of lung cancer. Collectively, expression loss or mutations compromising XRN2 function could promote DNA damage, elevate replication stress, enhance genomic instability, and contribute in carcinogenesis. Thus, understanding the cellular function of XRN2 in processes beyond RNA metabolism is critical to derive a mechanistic understanding of complex phenotype arising after XRN2 loss and identify potential avenues for targeting XRN2 vulnerabilities in cancer. Our preliminary data show that XRN2 associates with PARP1, however, the significance of this interaction remains unknown. Through our specific aims, we will delineate the molecular basis of interplay between XRN2 and PARP1, and develop the mechanistic understanding of how the interaction between these two protein modulate their enzymatic functions. Collectively, this work will provide the fundamental knowledge of strategies cells employ to prevent R-loop-induced genomic instability and how XRN2-associated vulnerabilities could be targeted against cancer.